Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing

Abstract

Endosomal escape remains a fundamental barrier hindering the advancement of nucleic acid therapeutics. Taking inspiration from natural phospholipids that comprise biological membranes, we report the combinatorial synthesis of multi-tailed ionizable phospholipids (iPhos) capable of delivering messenger RNA or mRNA/single-guide RNA for gene editing in vivo. Optimized iPhos lipids are composed of one pH-switchable zwitterion and three hydrophobic tails, which adopt a cone shape in endosomal acidic environments to facilitate membrane hexagonal transformation and subsequent cargo release from endosomes. Structure-activity relationships reveal that iPhos chemical structure can control in vivo efficacy and organ selectivity. iPhos lipids synergistically function with various helper lipids to formulate multi-component lipid nanoparticles (called iPLNPs) for selective organ targeting. Zwitterionic, ionizable cationic and permanently cationic helper lipids enable tissue-selective mRNA delivery and CRISPR-Cas9 gene editing in spleen, liver and lungs (respectively) following intravenous administration. This rational design of functional phospholipids demonstrates substantial value for gene editing research and therapeutic applications.

Fig. 1 ǀ. A combinatorial library of iPhos lipids was chemically synthesized and studied, which led to the elucidation of a physical mechanism of action for enhanced endosomal escape.

a, Efficacious iPhos lipids were composed of one ionizable amine, one phosphate group, and three hydrophobic alkyl tails. Upon entering acidic endosomes/lysosomes, protonation of the tertiary amine induced a zwitterionic head group, which could readily insert into membranes. b, Most biological membrane phospholipids possess a zwitterion and adopt a lamellar phase. When iPhos lipids were mixed and inserted into the endosomal membranes, the formed cone shape by small ion pair head and multiple hydrophobic tails enabled hexagonal transformation. c, Synthetic routes of iPhos: alkylated dioxaphospholane oxide molecules (Pm) were conjugated to amines (nA) to obtain iPhos (nAxPm). “x” in “nAxPm” indicated the number of Pm molecules modified on one amine molecule. d, A list of 28 amines nA and 13 alkylated dioxaphospholane oxide Pm molecules used for iPhos synthesis.

Fig. 2 ǀ. Structure-activity relationships of iPhos lipids for luciferase mRNA delivery in vitro.

a, A heat map of luciferase expression following treatment of IGROV1 cells with iPLNPs (50 ng firefly luciferase (Fluc) mRNA, n = 3 biologically independent samples). RLU > 10,000 was counted for the hit rate calculation. b, Representative chemical structures of iPhos with different numbers of zwitterions and tails in an acidic endosomal environment. c, The relative hit rate of iPhos with a single zwitterion and multiple zwitterions. d, The relative hit rate of iPhos (1A1P4–18A1P16) with a single zwitterion and different numbers of tails. e, Among the efficient iPhos (7A1P4–13A1P16), tail length of starting amines influenced the ultimate in vitro efficacy.

Fig. 3 ǀ. Model membrane studies of endosomal escape demonstrated the mechanism of iPhos lipid-mediated RNA delivery with correlation to chemical structure.

a, ³¹P NMR spectra of endosomal mimic and a mixture of endosomal mimic with iPhos 9A1P9. iPhos lipid mixing induced membrane hexagonal H_II transformation. b, Hemolysis of 17A and 10A1P10 at pH 5.5. The zwitterion could significantly benefit the endosomal escape. c, Hemolysis of 9A1P9 and 10A1P10 at different pHs. d, Hemolysis of iPLNPs at different pHs. e, Lipid fusion and membrane rupture of 10A1P10 and iPLNPs were determined by a FRET assay at pH 5.5. f, iPLNP dissociation by FRET characterization after mixing with anionic endosomal mimics for 10 min at pH 5.5. A single zwitterion showed higher lipid fusion and iPLNP dissociation efficacy than multiple zwitterions. g, 10A1P10 iPLNP dissociation of different time intervals at pH 5.5. Data in b-g are presented as mean ± s.d. (n = 3 biologically independent samples). Statistical significance was analyzed by the two-tailed unpaired t-test: ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Fig. 4 ǀ. Structure-activity studies revealed that iPhos lipid structure controlled in vivo efficacy and organ selectivity.

a, In vivo evaluation of 51 iPhos at a low Fluc mRNA dose (0.1 mg kg⁻¹). Bioluminescence images of various organs were recorded 6 h after IV injection of iPLNPs into C57BL/6 mice. H: Heart, Lu: lung, Li: Liver, K: Kidney, S: Spleen. b, Among the efficacious 10A1P4–12A1P16 iPhos, hydrophobic chain length at the amine side significantly influenced the in vivo mRNA delivery efficacy. c, mRNA expression in liver by iPhos with different alkyl chain length at phosphate group side. 9 to 12 carbon lengths were the most efficient. d, mRNA expression in spleen by iPhos with different alkyl chain length at phosphate group side. 13 to 16 alkyl chain lengths were the most efficient.

Fig. 5 ǀ. iPhos outperformed traditional phospholipids, and functioned with different helper lipids for organ selective RNA delivery.

a, Structure of iPhos 9A1P9 in the acidic endosomal environment. b,c, iPhos 9A1P9 outperformed benchmark DOPE and DSPC for mRNA delivery. Images (b) and quantification (c) of luciferase expression in liver were recorded (Fluc mRNA, 0.25 mg kg⁻¹). H: Heart, Lu: lung, Li: Liver, K: Kidney, S: Spleen. d,e, iPLNPs containing zwitterionic helper lipid DOPE mediated mRNA expression in spleen. In vivo evaluation (d) and quantification (e) were evaluated (Fluc mRNA, 0.25 mg kg⁻¹). f,g, iPLNPs containing ionizable cationic helper lipids mediated mRNA translation in liver. Organ selectivity (f) and quantification (g) of Fluc mRNA expression by 9A1P9 iPLNP with different ionizable cationic helper lipids were assayed (Fluc mRNA, 0.25 mg kg⁻¹ for MDOA and DODAP; 0.05 mg kg⁻¹ for 5A2-SC8). h,i, iPLNPs containing permanently cationic helper lipids induced mRNA transfection in lung. Organ images (h) and quantification (i) of Fluc mRNA expression by 9A1P9 iPLNP using DDAB and DOTAP were evaluated (Fluc mRNA, 0.25 mg kg⁻¹). j,k, 9A1P9–5A2-SC8 iPLNP showed much higher mRNA delivery efficacy than positive control DLin-MC3-DMA LNPs. Images (j) and quantification (k) of luciferase expression in liver were recorded (Fluc mRNA, 0.05 mg kg⁻¹). l-n, 9A1P9 iPLNPs enabled Cre mRNA delivery selectively in liver or lung. Schematic (l) represented a Cre-LoxP mouse model that could express tdTomato by translating Cre-recombinase mRNA to Cre protein to delete the stop. 9A1P9–5A2-SC8 iPLNP (m) and 9A1P9-DDAB iPLNP (n) mediated tdTomato expression in liver and lung, respectively (Cre mRNA, 0.25 mg kg⁻¹). All data are presented as mean ± s.d. (n = 3 biologically independent mice). Statistical significance was analyzed by the two-tailed unpaired t-test: ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Fig. 6 ǀ. iPLNPs enabled CRISPR/Cas9 gene editing selectively in liver and lungs and possessed potential for clinical translation.

a, Schematic of co-delivery of Cas9 mRNA and sgTom1 deletes the stop cassettes and activates tdTomato protein. b, 9A1P9–5A2-SC8 iPLNPs enabled gene editing specifically in liver. c, 9A1P9-DDAB iPLNPs enabled gene editing specifically in lung. d, Following IV administration of 9A1P9–5A2-SC8 iPLNPs containing Cas9 mRNA and sgTom1 to Ai9 mice, tdTomato-positive cells were observed in liver. Scale bars, 50 μm. e, Confocal fluorescence images showed tdTomato-positive cells in lung after administration of 9A1P9-DDAB iPLNPs. Scale bars, 50 μm. f, T7E1 assay of organ selective gene editing. 9A1P9–5A2-SC8 and 9A1P9-DDAB iPLNPs containing Cas9 mRNA and sgPTEN were IV administered into C57BL/6 mice, enabling CRISPR/Cas9 gene editing in liver and lung, respectively. For all the CRISPR/Cas9 gene editing assays, Cas9 mRNA/sgRNA weight ratio of 4:1 and total RNA dose of 0.75 mg kg⁻¹ were used. g,h, iPLNPs were prepared by controlled microfluidic mixing, which resulted in decreased iPLNP sizes and preserved efficacy and organ selectivity. 9A1P9–5A2-SC8 iPLNPs (liver specific, Fluc mRNA, 0.05 mg kg⁻¹) (g) and 9A1P9-DDAB iPLNPs (lung specific, Fluc mRNA, 0.25 mg kg⁻¹) (h) demonstrated small sizes and fully retained precise organ selectivity. i,j, 9A1P9–5A2-SC8 iPLNPs (Fluc mRNA, 0.05 mg kg⁻¹) allowed repeat dosing without loss of efficacy. Whole body imaging (i) and quantification of luciferase expression (j) was performed 6 h after each injection. k, 9A1P9–5A2-SC8 and 9A1P9-DDAB iPLNPs were well tolerated in vivo. Data are presented as mean ± s.d. and statistical significance was analyzed by the two-tailed unpaired t-test: ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; NS, P > 0.05. All data are from n = 3 biologically independent mice.